Stereoselectivity of each of the three steps of the heme oxygenase reaction: hemin to meso-hydroxyhemin,meso-hydroxyhemin to verdoheme,and verdoheme to biliverdin

Heme oxygenase catalyzes the regiospecific oxidation of hemin to biliverdin IXalpha with concomitant liberation of CO and iron by three sequential monooxygenase reactions. The alpha-regioselectivity of heme oxygenase has been thought to result from the regioselective oxygenation of the heme alpha-meso position at the first step, which leads to the reaction pathway via meso-hydroxyheme IXalpha and verdoheme IXalpha intermediates. However, recent reports concerning heme oxygenase forming biliverdin isomers other than biliverdin IXalpha raise a question whether heme oxygenase can degrade meso-hydroxyhemin and isomers other than the alpha-isomers. In this paper, we investigated the stereoselectivity of each of the two reaction steps from meso-hydroxyhemin to verdoheme and verdoheme to biliverdin by using a truncated form of rat heme oxygenase-1 and the chemically synthesized four isomers of meso-hydroxyhemin and verdoheme. Heme oxygenase-1 converted all four isomers of meso-hydroxyhemin to the corresponding isomers of verdoheme. In contrast, only verdoheme IXalpha was converted to the corresponding biliverdin IXalpha. We conclude that the third step, but not the second, is stereoselective for the alpha-isomer substrate. The present findings on regioselectivities of the second and the third steps have been discussed on the basis of the oxygen activation mechanisms of these steps.     

Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X(2) receptor

P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na(+), these channels also conduct a significant Ca(2+) current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis of this Ca(2+) conductance by a mutational analysis of recombinant P2X(2) receptors. Wild type and 31 mutant P2X(2) receptors were expressed in HEK-293 cells and studied under voltage-clamp. We found that the relative Ca(2+) permeability measured from the reversal potentials of ATP-gated currents was unaffected by neutralizing fixed charge (Asp(315), Asp(349)) near the mouths of the channel pore. By contrast, mutations that changed the character or side chain volume of three polar residues (Thr(336), Thr(339), Ser(340)) within the pore led to significant changes in P(Ca)/P(Cs). The largest changes occurred when Thr(339) and Ser(340) were replaced with tyrosine; these mutations almost completely abolished Ca(2+) permeability, reduced P(Li)/P(Cs) by about one-half, and shifted the relative permeability sequence of Cs(+), Rb(+), K(+), and Na(+) to their relative mobility in water. Our results suggest that the permeability sequence of the P2X(2) receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain.     

Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice

Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum+dexamethasone+ascorbate-2-phosphate+beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+1% fetal bovine serum+insulin+ascorbate-2-phosphate+transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering.     

RORalpha1 and RORalpha4 suppress TNF-alpha-induced VCAM-1 and ICAM-1 expression in human endothelial cells

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) is a nuclear orphan receptor. Adenovirus-mediated overexpression of RORalpha1 and RORalpha4 suppressed tumor necrosis factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells. Overexpression of RORalpha1 and RORalpha4 also suppressed TNF-alpha-stimulated translocation of p50 and p65 to the nucleus. In contrast, dominant-negative deletion mutants of RORalpha1 and RORalpha4 failed to suppress the induction of VCAM-1 and ICAM-1 and translocations of p50 and p65. These results suggest that RORalpha1 and RORalpha4 regulate the inflammatory responses via inhibition of the nuclear factor-kappaB signaling pathway in endothelial cells.     

Cloning and characterization of two novel zebrafish P2X receptor subunits

Activation of Kupffer cells by lipopolysaccharide (LPS) after ethanol feeding results in overproduction of TNF-alpha, leading to liver injury. Since dilinoleoylphosphatidylcholine (DLPC) protects against liver injury and has antioxidant properties, we investigated whether it alters LPS signaling leading to decreased TNF-alpha production. Kupffer cells were isolated from rats fed alcohol-containing or isocaloric control diets for 3 weeks. With ethanol, cytochrome P4502E1 was upregulated. When stimulated with LPS in culture, Kupffer cells released more TNF-alpha compared to control rats; DLPC diminished the increase. It also reduced ERK1/2 and p38 phosphorylation as well as NF-kappaB activation with decreased nuclear p65 and increased cytosolic IkappaB-alpha expression. ERK1/2 and NF-kappaB activation were abolished by the ERK1/2 inhibitor PD098059. The p38 inhibitor SB203580 abolished p38 activation without affecting NF-kappaB. Both inhibitors reduced TNF-alpha generation. Thus, DLPC diminishes LPS-dependent TNF-alpha generation by inhibiting p38 and ERK1/2 activation; the latter leads to decreased NF-kappaB activation.     

Metabolism of Threonine,Methionine, and Related Compounds in Mixed Rumen Ciliate Protozoa1

Metabolic pathways of L-threonine and L-methionine in starved, bacteria-free, mixed rumen ciliate protozoa were examined. Rumen ciliates produced 2-oxobutanoate (2OB) from both L-threonine and L-methionine; 2OB was then converted to 2-aminobutanoate (2AB) and propionate. The 2AB appeared to be converted slowly to propionate. D-Threonine seemed to be hardly metabolized.     

TheH-2 class II genes and the susceptibility to the development of pulmonary adenoma in mice

To determine the locus in theH-2 complex that affects susceptibility to the development of pulmonary adenomas in mice,H-2 congenic and recombinant strains of mice with A/Wy, BALB/c, C3H, and B10 backgrounds were subjected to treatment with urethane. The average number and the incidence of adenoma foci were recorded five months after the treatment. InH-2 congenic strains on the A/Wy background, the average number of adenoma foci per mouse was significantly higher in mice of the A/Wy, A/J, and A-Tla ^b (H-2 ^a ) strains than in A.BY (H-2 ^b ) mice. In BALB/c and C3H congenic strains, the strains carrying theH-2 ^k haplotype were more susceptible than those carrying theH-2 ^b haplotype. InH-2 congenic strains on the B 10 background, the average number and incidence of foci was also higher in haplotypesa, h2, k, andj than in haplotypesb, s, f, d, r, h4, i3, i5, and4. The average numbers of adenoma foci in (A/J × A.BY)F₁ (H-2 ^a /H-2 ^b ) and (B10 × B10.A)F₁ (H-2 ^b /H-2 ^a ) were intermediate between the numbers in the parental strains. In [B10.A (4R) × B10.A (3R)]F₁ (H-2 ^h4 /H-2 ⁱ³ ) and [B10.A (4R) × B10.A (5R)]F₁ (H-2 ^h4 /H-2 ⁱ⁵ ), the numbers of adenoma foci were higher than in resistant parental recombinants. These patterns of response to urethane matched the patterns of the immune response to lactate dehydrogenase-B (LDH-B) and immunoglobulin gamma 2a (IgG2a) proteins. These differences between mice in their susceptibility to the development of pulmonary adenomas is probably due to the polymorphism of the class II genes in theH-2 complex.     

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross- reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand - Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.      

Synergistic effects of phorbol ester and interferon-alpha: target cell class I HLA antigen expression and resistance to natural killer and lymphokine-activated killer cell-mediated cytolysis

This study was undertaken to investigate whether target cell class I HLA antigen expression induced by phorbol ester and interferon-alpha (IFN-alpha) was associated with resistance to natural killer (NK) cells and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Class I antigen expression on the surface of the K562 erythroleukemia cell line was enhanced by either IFN-alpha or phorbol ester (PDBu). Addition of PDBu together with IFN-alpha had a synergistic effect on class I antigen expression on the cells. Furthermore, synergism between IFN-alpha and PDBu was also found in class I antigen expression by MOLT-3 cells. This synergistic effect on class I antigen expression was blocked by the protein synthesis inhibitor (cycloheximide). Pretreatment of K562 cells with PDBu and IFN-alpha made them more resistant to lysis by NK and LAK cells than did either PDBu or IFN-alpha. In contrast to PDBu, 4 alpha PDD, a biologically inactive phorbol analogue, alone or combination with IFN-alpha, had no effect on class I antigen expression and susceptibility to lysis by NK and LAK cells. Kinetic experiments showed an inverse relationship between the expression of class I antigens and susceptibility to NK cell-mediated cytolysis. Using cold target competition analysis, target cells pretreated with PDBu and IFN-alpha clearly competed less effectively than did untreated cells for lysis of untreated target cells. These results demonstrate that target cells pretreated with PDBu and IFN-alpha decrease their sensitivity to natural killer and lymphokine-activated killer cells inversely with target cell class I HLA antigen expression.